This invention relates to a process for the production of stable, lyophilized erythrocyte preparations sensitized by coating with an antigen or antigen-protein conjugate, respectively which are useful for the quantitative determination of the antigen, e.g., in hemagglutination reactions. More particularly, the process of this invention is characterized by combining erythrocytes, antigen, glutaraldehyde and formaldehyde in the presence of a buffer solution, allowing the components to simultaneously act on each other under continuous agitation, washing and subsequently lyophilizing the thus-coated erythrocytes. This invention furthermore relates to the erythrocyte preparation obtained according to this process.
Red blood cells or erythrocytes (generally from sheep) are widely used in immunochemical diagnostic techniques, particularly sensitized erythrocytes to which an immunologically or biochemically active group has been directly or indirectly bonded for hemagglutination testing. In virtually all diagnostic procedures employing sensitized erythrocytes, the freshness of the erythrocytes themselves as well as of the sensitized preparation has been a key factor necessary to obtain accurate and reproducible test results. While the preparation of sensitized erythrocyte complexes requires materials, time and skills not always available in clinical laboratories, the instability of such preparations has severely limited commercial exploitation of the potential broad use for such materials manufactured on a large scale.
The problems encountered in freezing red blood cells are compounded when using sensitized erythrocytes. Not only must cell wall rupture be minimized to the greatest extent possible, but this must be done without adversely affecting the sensitization coating as well.
Suitable antigens known to be useful in sensitizing erythrocytes include but are not limited to the proteohormones, e.g., corpus luteam hormone (LH, luteinizing hormone), follicle-stimulating hormone (FSH), growth hormone (STH, somatotropic hormone), prolactin, insulin, etc., and in particular human chorionic gonadotrophin (HCG) and human placenta lactogen (HPL). As these latter two proteohormones in particular are found in the blood and urine of pregnant women, the quantitative determination thereof is of great importance in the detection and monitoring of a pregnancy. L. Wide and C. A. Gemzell developed pregnancy reactions with antigen-bound erythrocytes and antiserum described in Acta Endocrinol. 35: 261 (1960). These reactions are useful both for monitoring pregnancies and for the early determination of pathological manifestations.
Enzymes are also suitable as antigens for sensitizing erythrocytes, e.g., glutamate-oxalacetate transaminase (GOT), glutamic pyruvic transaminase (GPT), lactate dehydrogenase, etc.
Useful antigens also include a great variety of different substances, the detection of which in the blood, urine or other body fluids by techniques employing sensitized erythrocytes is of clinical interest. Among these substances are peptide hormones, e.g., oxytocin and angiotensin; steroid hormones, e.g., testosterone, estradiol, hydrocortisone, etc.; medicinal agents and drugs, e.g., fluocortolone, lysergic acid diethylamide, etc. It will be readily apparent that rapid quantitative determinations of all these substances for diagnostic purposes and for control or screening examinations are of great value to clinicians.
Substances having a low molecular weight can be bound to the erythrocytes directly, if they possess functional groups suitable for cross-linking, e.g., amino groups, or after the introduction of such groups, for use in the process of this invention. Since this binding process often causes damage to the erythrocytes, they can advantageously be first linked with a high-molecular weight "carrier" protein, which serves to protect the substance of interest from causing excessive damage to the erythrocytes during coupling. The linking process resides in the formation of a chemical bond between the antigen and the carrier protein to form an antigen-protein conjugate, which is then coupled to the erythrocytes. Suitable carrier proteins are known and include but are not limited to albumins of various sources, e.g., bovine serum albumin and rabbit serum albumin; globulins of various species, e.g., bovine-.alpha.-globulin (BGG) and equine-.alpha.-globulin; synthetic polypeptides, e.g., poly-DL-Ala-poly-(Glu, Try)-poly-Lys and poly-(Glu,Tyr)-poly-DL-Ala-poly-Lys; etc.
Detection reactions with antigen-coated erythrocytes, or erythrocytes coated with antigen-protein conjugate, and antiserum are effected, e.g., by direct or indirect hemagglutination reactions analogously to the pregnancy determining and monitoring reactions, described above, as is known to those skilled in the art e.g., by direct and indirect hemagglutination (HA), hemagglutination inhibition (HAI), etc.
Antigen-coated erythrocytes have been described, e.g., see L. Wide, C. A. Gemzell, Acta Endocrinol. 35(1960) 261. However, the coated erythrocytes are unstable. Several attempts have been made to overcome this disadvantage by the use of coupling and stabilizing agents in the production of the erythrocyte preparations.
Thus, many bivalent reagents have been proposed as coupling agents between antigens and erythrocytes, e.g., bis-diazotized benzidines described in Int. Arch. Allergy 13:1 (1958); 1,3-difluoro-4,6-dinitrobenzene described in Immunology 4:49 (1961); toluene-2,4-diisocyanates described in Immunochemistry 1:43 (1964); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide described in J. Immunol. 97:791 (1966); glutaraldehyde described in Brit. J. Haemat. 7:299 (1961); etc. Since even the coupling-coated erythrocytes are still readily susceptible to decomposition, the erythrocytes have further been hardened by treating with formaldehyde prior to coating, e.g., using the procedure described in Proc. Soc. Exp. Biol. (N.Y.) 99:452 (1958). The processes are thereby made complicated and expensive and, due to the numerous stages employed, do not always yield reproducible results.
In DOS (German Unexamined Laid-Open Application) 2,132,499, a serum-diagnostic composition of chorionic gonodotrophin bound to red blood cells by means of glutaraldehyde as the coupling agent is described. In order to increase the stability of the sensitized erythrocytes, these blood cells are aftertreated with glutaraldehyde or formaldehyde. The sensitized blood cells are obtained in the form of a suspension in a buffer solution. The aftertreatment with glutaraldehyde or formaldehyde renders the process expensive; furthermore, the HCG-sensitized red blood cells can be satisfactorily preserved in the suspension only if the latter is stored under refrigeration at 2.degree.-8.degree. C.